转录因子Krüppel样因子4抑制骨髓间充质干细胞向髓核样细胞分化研究  

Transcription factor Krüppel-like factor 4 inhibits the differentiation of bone marrow-derived mesenchymal stem cells to nucleus pulposus-like cells

作　　者：王亚寒[1] 罗建平[1] 王小刚[1] 杨彬[1] 张新胜 Wang Yahan;Luo Jianping;Wang Xiaogang;Yang Bin;Zhang Xinsheng(Department of Orthopedics,People's Hospital of Henan Province,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院骨科,郑州450003

出　　处：《中华实验外科杂志》2019年第1期27-30,共4页Chinese Journal of Experimental Surgery

摘　　要：目的 观察Krüppel样因子4(KLF4)对转化生长因子(TGF)-β1诱导的人骨髓间充质干细胞(BMSCs)向髓核样细胞分化的影响。方法实时定量聚合酶链反应(Real-time PCR)和Western blot检测TGF-β1诱导BMSCs 7 d和14 d对KLF4 mRNA和蛋白表达的影响;建立KLF4过表达BMSC细胞株,用TGF-β1诱导7 d或14 d,细胞计数试剂盒(CCK-8)检测细胞增殖,Real-time PCR和Western blot分析髓核细胞相关蛋白聚集蛋白聚糖(Aggrecan)、Ⅱ型胶原蛋白α1链(Col2A1)、性别决定区Y框蛋白9(SOX9)、角蛋白19(KRT19)和叉头蛋白(FOXF1)表达。结果 与对照组比较,TGF-β1诱导后KLF4 mRNA和蛋白表达均显著降低(第7天mRNA:0.653±0.083比1.027±0.068,t=6.012,P<0.01;第14天mRNA:0.453±0.065比1.063±0.096,t=9.105,P<0.01;第7天蛋白,0.601±0.112比0.873±0.051,t=3.874,P<0.05;第14天蛋白:0.251±0.087比0.893±0.078,t=9.543,P<0.01),细胞增殖增加(第7天,166.355±11.132比99.647±6.220,t=9.064,P<0.01;第14天:195.333±10.512比124.667±9.609,t=8.598,P<0.01),Aggrecan、Col2A1、SOX9、KRT19、FOXF1 mRNA (Aggrecan:2.600±0.255比1.017±0.095,t=10.07,P<0.01;Col2A1:3.441±0.259比1.010±0.076,t=15.58,P<0.01;SOX9:2.767±0.139比1.033±0.111,t=16.92,P<0.01;KRT19:3.710±0.210比1.005±0.076,t=20.95,P<0.01;FOXF1:2.783±0.174比1.035±0.132,t=13.88,P<0.01)和蛋白(Aggrecan:0.760±0.072比0.233±0.055,t=10.05,P<0.01;Col2A1:0.941±0.046比0.190±0.040,t=21.36,P<0.01;SOX9:1.237±0.140比0.317±0.065,t=10.31,P<0.01;KRT19:0.897±0.072比0.137±0.051,t=14.84,P<0.01;FOXF1:1.033±0.191比0.181±0.056,t=7.434,P<0.01)表达升高。与TGF-β1单独处理比较,过表达KLF4抑制了细胞增殖(第7天:125.750 ±10.386比166.355±11.132,t=4.620,P<0.05;第14天:148.526±13.449比195.333±10.512,t=4.702,P<0.01),降低了Aggrecan、Col2A1、SOX9、KRT19、FOXF1 mRNA (Aggrecan:1.510±0.187比2.600±0.255,t=5.970,P<0.01;Col2A1:1.727±0.254比3.441±0.259,t=8.176,P<0.01;SOX9:1.493±0.169比2.767±0.139,t=10.08,P<0.01;KRT19:2.117±0.387比3.710±0.210,t=6.273,P<0.01;FOXFObjective To investigate the effect of Krüppel-like factor 4 (KLF4) on the differentiation of human bone marrow-derived mesenchymal stem cells (BMSC) to nucleus pulposus-like cells induced by transforming growth factor (TGF)-β1.Methods Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to assay the effects of TGF-β1 on the mRNA and protein levels of KLF4 on days 7 and 14. Then, TGF-β1 was used to stimulate KLF4-overexpressing cell line for 7 days or 14 days. Cell proliferation was analyzed by cell counting kit-8 (CCK-8). Real-time PCR and Western blotting were used to detect the mRNA and protein levels of nucleus pulposus-related proteins Aggrecan, collagen type Ⅱ α1 (Col2A1), sex determining region Y box protein 9 (SOX9), keratin 19 (KRT19) and forkhead box F1 (FOXF1).Results Compared with the control group, TGF-β1 notably reduced the mRNA and protein levels of KLF4 (Day 7 mRNA: 0.653±0.083 vs. 1.027±0.068, t=6.012, P<0.01;Day 14 mRNA: 0.453±0.065 vs. 1.063±0.096, t=9.105, P<0.01;Day 7 protein: 0.601±0.112 vs. 0.873±0.051, t=3.874, P<0.05;Day 14 protein: 0.251±0.087 vs. 0.893±0.078, t=9.543, P<0.01), promoted cell proliferation (Day 7: 166.355±11.132 vs. 99.647±6.220, t=9.064, P<0.01;Day 14: 195.333±10.512 vs. 124.667±9.609, t=8.598, P<0.01), and elevated the mRNA (Aggrecan: 2.600±0.255 vs. 1.017±0.095, t=10.07, P<0.01;Col2A1: 3.441±0.259 vs. 1.010±0.076, t=15.58, P<0.01;SOX9: 2.767±0.139 vs. 1.033±0.111, t=16.92, P<0.01;KRT19: 3.710±0.210 vs. 1.005±0.076, t=20.95, P<0.01;FOXF1: 2.783±0.174 vs. 1.035±0.132, t=13.88, P<0.01) and protein (Aggrecan: 0.760±0.072 vs. 0.233±0.055, t=10.05, P<0.01;Col2A1: 0.941±0.046 vs. 0.190±0.040, t=21.36, P<0.01;SOX9: 1.237±0.140 vs. 0.317±0.065, t=10.31, P<0.01;KRT19: 0.897±0.072 vs. 0.137±0.051, t=14.84, P<0.01;FOXF1: 1.033±0.191 vs. 0.181±0.056, t=7.434, P<0.01) levels of Aggrecan, Col2A1, SOX9, KRT19 and FOXF1. Compared with TGF-β1-treated only group, KLF4 overexpression significantly inhibite

关 键 词：Krüppel样因子4 骨髓间充质干细胞 髓核细胞 增殖 分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]